Although screening mammography and the increased use of breast conserving surgery and adjuvant chemotherapy have improved the quality of life and prolonged survival for women with breast cancer, additional therapeutic strategies are needed to combat the disease. Various studies have suggested dietary fat, especially polyunsaturated fatty acids, promotes tumor growth by increasing synthesis of eicosanoids, particularly arachidonic acid (AA) products. The possible role of AA-derived eicosanoids as regulators of neoplastic cell growth is an area of significant interest in breast cancer biology.
Digital Rectal Examination (DRE) and Prostate Specific Antigen (PSA) tests are routinely used to screen for the presence of prostate cancer in men. However, there is a serious debate in the medical community as to whether DRE and/or PSA screening accurately predict the presence of prostate cancer. For instance, the American Cancer Society now recommends that physicians inform men screened for prostate cancer that a PSA result of less than 4.0 ng/ml does not guarantee that cancer is not present, because up to 25% of men with the disease can have PSA levels of under 4.0 ng/ml. Furthermore, an “abnormal” or elevated PSA can be caused by benign growth, inflammation, or other causes. Therefore, a number of men with elevated PSA levels may require additional diagnostic tests and in the end, some may not require treatment.
Epidemiological studies on carcinoma of the prostate gland have shown a positive relationship between the consumption of dietary fats and development of prostate cancer. (Franceschi S.: Fat and prostate cancer. Epidemiol 5:271–273, 1994.; Snowdon D A, Phillips R L and Choi W.: Diet obesity and risk of fatal prostatic cancer. Amer J Epidemiol 2:244–250, 1984). This led to the suggestion that high dietary fat intake may be a contributing factor in the initiation or development of this tumor (Wynder E L, Laakso K, Sotarauta M and Rose D.: Metabolic epidemiology of prostatic cancer. Prostate 5:47–53, 1984). Conversely, (Wang U, Corr J G, Thaler H T, Tao Y, Fair W R and Heston W D W.: Decreased growth of established human prostate LNCaP tumors in nude mice fed a low-fat diet. J. Nat Cancer Inst 87:1456–1462, 1995) showed that lowering the quantity of fat as a proportion of total calories decreased the growth rate of human prostate adenocarcinoma cells in mice.
Studies have implicated an association of linoleic acid (LA), a constituent of dietary fat, with prostate cancer. Linoleic acid is the most prevalent unsaturated fatty acid component of commonly used cooking oils. A large prospective study of American men showed a positive association between linoleic acid in the diet and prostate cancer. (Giovannucci E, Rimm E B, Colditz G A, Stampfer M J, Ascherio A, Chute C C and Willett W C.: A prospective study of dietary fat and risk of prostate cancer. J Nat Cancer Inst 85:1571–1579, 1993). Moreover, in vitro studies of the human prostate cancer cell line PC-3 showed stimulated growth in the presence of linoleic acid whereas the long chain fatty acids may inhibit tumorigenesis. (Rose DP and Connolly J M.: Effects of fatty acids and eiconsanoid synthesis inhibitors on the growth of two human prostate cancer cell lines. Prostate 1811243–254:1991). Recently Harvei et al. showed an association of serum levels of linoleic acid and palmitic acid with increased risk of prostate cancer. (Harvei S, Bjerve K S, Tretli S, Jellum E, Robsahm T E and Vatten L.: Prediagnostic level of fatty acids in serum phospholipids: Ω-3 and Ω-6 fatty acids and the risk of prostate cancer. Int J Cancer 71:545–551, 1997).
There are no current serum tests available for breast cancer. The present screening tests for breast and prostate cancer do not take into account the presence of fats or fat-metabolizing substances in body tissue or fluids. There exists a need for improved methods of diagnosing and treating cancer, particularly breast and prostate cancer. There further exists a need for a composition of matter for use in the treatment of cancer, particularly breast and prostate cancer.
It has been shown that there is a positive correlation between high dietary fat and development of breast cancer. Thus, it appears that arachidonic acid acts as a potent mitogen for human breast cancer cells. FABPs bind fatty acids noncovalently with high affinity and translocates them across the cell to the nuclear receptors. The existence of various FABP types and the relative abundance of these cytoplasmic proteins in nearly all tissues indicate important functions for these molecules.
AA and its metabolites are well known to bind to the FABP with high affinity in liver carcinogenesis. The expression of one of the FABPs, designated L-FABP, has been shown to be upregulated in liver during carcinogenesis. (Custer R P and Sorof S.: Target polypeptide of a carcinogen is associated with normal mitosis and carcinogen-induced hyperplasias in adult hepatocytes. Proc Natl Acad Sci USA 81:7638–6742, 1984. Custer R P and Sorof S.: Mitosis in hepatocytes is generally associated with elevated levels of the target polypeptide of a liver carcinogen. Differentiation 30:176–181, 1985). Furthermore, secreted L-FABP has been detected in serum of rats with hepatocarcinoma. Only one previous study addressed the issue of FABPs in prostate cancer (Chaudry A A and Dutta-Roy A K.: Purification and characterization of a fatty acid binding protein from human prostatic tissue. Lipids 28:383–8, 1993.). That study suggested that an L-FABP-like protein occurred in both normal and cancerous prostate cells. Other FABP types have never been identified in prostate gland and have never been implicated in control of cell proliferation or cancer. The levels of L-FABP has been shown to increase in liver carcinogenesis compared to the normal tissue and L-FABP is also known to be secreted in the serum, however nothing is known about the levels of FABPs in different stages of breast cancer, or the effect of hormones, growth factors or bioactive lipids on FABPs.
Certain FABPs have been reported to have differential effects on cell growth when cDNA clones have been transfected into these cells. Transfection of L-FABP into hepatoma cells increased proliferation (Keler T, Barker C S and Sorof S.: Specific growth stimulation by linoleic acid in hepatoma cell lines transfected with the target protein of a liver carcinogen. Proc Natl Acad Sci USA 89:4830–4, 1992; Keler T and Sorof S.: Growth promotion of transfected hepatoma cells by liver fatty acid binding protein. J Cell Physiol 157:33–40, 1993; Sorof S.: Modulation of mitogenesis by liver fatty acid binding protein. Cancer Metastasis Rev 13:317–36, 1994.). In contrast, MDGI (H-FABP) appears only in normal and not tumor mammary cells (Grosse R, Boehmer F D, Langen P, Kurtz A, Lehmann W, Mieth M and Wallukat G.: Purification, biological assay and immunoassay of mammary-derived growth inhibitor. Methods Enzymol 198:425–440, 1991; Grosse R and Langen P.: Mammary derived growth inhibitor. In M. Sporn and A. Roberts, eds Handbook of Experimental Pharmacology. Heidelberg, 1990, pp 249–265.) and transfection of a cDNA clone of MDGI into breast cancer cells or mouse mammary epithelial cells results in loss of tumorigenicity (Huynh H, Alpert L and Pollak M.: Silencing of the mammary-derived growth inhibitor (MDGI) gene in breast neoplasms is associated with epigenetic changes. Cancer Res 56:4865–70, 1996.). FABPs are known to bind many different groups of fatty acids and their derivatives, including eicosanoids and other bioactive lipids, reviewed in (Veerkamp J H, Peeters R A and Matman R G H J.: Structural and functional features of different types of cytoplasmic fatty acid-binding proteins. Biochim Biophys Acta 1081:1–24, 1991). L-FABP exhibits different lipid binding characteristics from that of A-FABP or H-FABP. L-FABP transfected into rat hepatoma cells also mediates cell induction by carcinogenic peroxisome proliferators (Khan S H and Sorof S.: Liver fatty acid-binding protein: specific mediator of the mitogenesis induced by two classes of carcinogenic peroxisome proliferators. Proc Natl Acad Sci U S A 91:848–52, 1994.). Several studies suggest that FABP increases the solubility of fatty acids in the cell cytoplasm causing a net diffusion of fatty acids from the plasma membrane to the intracellular membrane compartments (Tipping E and Ketterer B.: The influence of soluble binding proteins on lipophile transport and metabolism in hepatocytes. Biochem J 195:441–52, 1981; Vork M M, Glatz J F C and Van Der Vusse G J.: On the mechanism of long chain fatty acid transport in cardiomyocytes as facilitated by cytoplasmic fatty acid-binding protein. J Theoret Biol 160:207–222, 1993.).
L-FABP is elevated significantly in metastatic or regenerating liver vs normal liver. This is in stark contrast to H-FABP, also known as mammary derived growth inhibitor (MDGI). It is present only in normal lactating breast, and completely disappears in mammary cancer cells (Grosse R, Boehmer F D, Langen P, Kurtz A, Lehmann W, Mieth M and Wallukat G.: Purification, biological assay and immunoassay of mammary-derived growth inhibitor. Methods Enzymol 198:425–440, 1991; Grosse R and Langen P.: Mammary derived growth inhibitor. In M. Sporn and A. Roberts, eds Handbook of Experimental Pharmacology. Heidelberg, 1990, pp 249–265). The inventors were intrigued by the differences in the effects on proliferation of MDGI vs L-FABP. The inventors performed a similar study (to the eicosanoid generation in hepatic cells) in MCF-7 cells transfected with a clone of the MDGI (H-FABP) gene or vector alone. The inventors also have examined these pairs of cells to determine cell cycle pattern changes related to MDGI (You Y., Zhang X., Das R. and Jett M. (1997) Cell cycle effects of mammary derived growth inhibitor in MDGI gene transfected breast cancer cells. In 37th Annual Meeting of American Society for Cell Biology, Abst #88.).
Changes in expression of FABPs have been reported for bladder cancer. Psoriasis-associated FABP (E-FABP) was noted to increase in level with increase in differentiation of bladder squamous cell carcinomas (Ostergaard M, Rasmussen H H, Nielsen H V, Vorum H, Orntoft T F, Wolf H and Celis J E.: Proteome profiling of bladder squamous cell carcinomas: identification of markers that define their degree of differentiation. Cancer Res 57:4111–7, 1997.). Although FABPs are intracellular proteins, H-FABP has been detected in elevated levels in plasma and urine of patients suffering from myocardial infarction, (Sohmiya K, Tanaka T, Tsuji R, Yoshimoto K, Nakayama Y, Hirota Y, Kawamura K, Matsunaga Y, Nishimura S and Miyazaki H.: Plasma and urinary heart-type cytoplasmic fatty acid-binding protein in coronary occlusion and reperfusion induced myocardial injury model. J Mol Cell Cardiol 25:1413–26, 1993; Van Nieuwenhoven F A, Kleine A H, Wodzig W H, Hermens W T, Kragten H A, Maessen J G, Punt C D, Van Dieijen M P, Van der Vusse G J and Glatz J F.: Discrimination between myocardial and skeletal muscle injury by assessment of the plasma ratio of myoglobin over fatty acid-binding protein. Circulation 92:2848–54, 1995; Wodzig K W, Kragten J A, Hermens W T, Glatz J F and van Dieijen-Visser M P.: Estimation of myocardial infarct size from plasma myoglobin or fatty acid-binding protein. Influence of renal function. Eur J Clin Chem Clin Biochem 35:191–8, 1997.) whereas psoriasis-associated FABP (E-FABP) was among a number of marker proteins detected in the urine of bladder cancer patients (Rasmussen H H, Orntoft T F, Wolf H and Celis J E.: Towards a comprehensive database of proteins from the urine of patients with bladder cancer. J Urol 155:2113–9, 1996.). In addition, loss of adipocyte-FABP (A-FABP) was reported with progression of human bladder transitional cell carcinomas (Celis J E, Ostergaard M, Basse B, Celis A, Lauridsen J B, Ratz G P, Andersen I, Hein B, Wolf H, Orntoft T F and Rasmussen H H.: Loss of adipocyte-type fatty acid binding protein and other protein biomarkers is associated with progression of human bladder transitional cell carcinomas. Cancer Res 56:4782–90, 1996.). The presence of A-FABP correlated with the grade and stage of the disease. The A-FABP protein was present in high levels in grade I and II TCCs whereas grade III had 37% reduction and grade IV had no A-FABP expression. A-FABP may act as a growth inhibitor similar to the MDGI (H-FABP) protein in breast cancer and loss of A-FABP expression may serve as a prognostic marker for aggressive bladder cancer.
Although FABPs are intracellular proteins, H-FABP has been detected in elevated levels in plasma and urine of patients suffering from myocardial infarction (Sohmiya K, Tanaka T, Tsuji R, Yoshimoto K, Nakayama Y, Hirota Y, Kawamura K, Matsunaga Y, Nishimura S and Miyazaki H.: Plasma and urinary heart-type cytoplasmic fatty acid-binding protein in coronary occlusion and reperfusion induced myocardial injury model. J Mol Cell Cardiol 25:1413–26, 1993; Van Nieuwenhoven F A, Kleine A H, Wodzig W H, Hermens W T, Kragten H A, Maessen J G, Punt C D, Van Dieijen M P, Van der Vusse G J and Glatz J F.: Discrimination between myocardial and skeletal muscle injury by assessment of the plasma ratio of myoglobin over fatty acid-binding protein. Circulation 92:2848–54, 1995; Wodzig K W, Kragten J A, Hermens W T, Glatz J F and van Dieijen-Visser M P.: Estimation of myocardial infarct size from plasma myoglobin or fatty acid-binding protein. Influence of renal function. Eur J Clin Chem Clin Biochem 35:191–8, 1997), whereas psoriasis-associated FABP (E-FABP) was among a number of marker proteins detected in the urine of bladder cancer patients (Rasmussen H H, Orntoft T F, Wolf H and Celis J E.: Towards a comprehensive database of proteins from the urine of patients with bladder cancer. J Urol 155:2113–9, 1996.). In addition, loss of adipocyte-FABP (A-FABP) was reported with progression of human bladder transitional cell carcinomas (Celis J E, Ostergaard M, Basse B, Celis A, Lauridsen J B, Ratz G P, Andersen I, Hein B, Wolf H, Orntoft T F and Rasmussen H H.: Loss of adipocyte-type fatty acid binding protein and other protein biomarkers is associated with progression of human bladder transitional cell carcinomas. Cancer Res 56:4782–90, 1996). The presence of A-FABP correlated with the grade and stage of the disease.
These results suggests that A-FABP and E-FABP may act as a tumor suppressors in prostate cells similar to MDGI (H-FABP) in MCF-7 cells (Huynh H, Alpert L and Pollak M.: Silencing of the mammary-derived growth inhibitor (MDGI) gene in breast neoplasms is associated with epigenetic changes. Cancer Res 56:4865–70, 1996.). Of the few FABPs tested so far, the inventors have found altered levels of FABPs; individually, each of these have been shown, in other cell systems, but not in breast or prostate to correlate with the normal or tumor state. The inventors are the first to show concomitant decreases in the heart-type FABPs (A-, H- and E-FABPs) and increases in mitosis promoting FABPs (I-, B- and L-FABP). These data support the notion that level(s) of FABP(s) will correlate with stage of prostate cell proliferation and perhaps aggressive tumors.
The inventors have shown for the first time the presence of multiple FABPs in different tissue types of cancer and have shown that the levels of the different FABPs are indicative of the presence and stage of cancer. For instance, the FABP message (L- and I-FABP) can be even 22 fold higher in tumor vs normal breast cells, especially in the estrogen receptor positive lines. The inventors have also discovered that the A-FABP, E-FABP class of proteins are downregulated in breast cancer cells. Prior to this study by the inventors, it has never been shown or suggested that the pattern of multiple forms of FABP play a significant role in carcinogenesis or that the presence of cancer can be diagnosed by measuring the level and types of FABPs present in a biopsy sample or serum sample.
The inventors have also discovered that free-radical scavengers called heteropolyanions (HPA), can effectively block the proliferation in cultures of breast tumor cells. Although HPAs were previously known, they were used for the treatment of HIV. The inventors have synthesized and identified free-radical scavengers, heteropolyanions, which effectively blocked proliferation in cultures of breast tumor cells. Prior to the inventors, no one had shown the effect of HPA on the growth of these breast cancer cells and the changes in the levels of these FAPB that play a role in carcinogenesis. No one has ever suggested treating cancer with an HPA or that HPA can affect the growth of breast or prostate cancer cells. Thus, FABPs also present a logical target for such intervention therapeutic drugs, which interrupt their crucial function of transmitting the cascades of signals of bioactive lipids that continually promote cancer cell proliferation.
The inventors have provided a basis for a better understanding of the direct role of fatty acids/bioactive lipids and FABPs in development and progression of breast cancer. FABPs can also be used as potential marker for breast cancer or, more specifically, for an aggressively growing breast cancer. There are no known detection markers for identification of breast cancer in a patient. The present invention can operate as a screening test for breast cancer.
It is therefore, an object of the present invention to provide a reliable method of diagnosing stage or aggressiveness of cancer, particularly breast and prostate cancer by measuring the presence and amounts of certain types of FABPs.
It is also an object of the present invention to provide a composition containing HPA in the form of a drug for treating cancer to block the cancer initiating function of certain FABPs.
These and other objects of the invention will become apparent from the following description.